Binding assay device

ABSTRACT

A device for performing an enzyme-labelled binding assay comprises an absorbent material and a developing solution, wherein the absorbent material is provided with a plurality of reagent zones including an indicator reagent zone, and is capable of transporting the developing solution by capillary action sequentially through each reagent zone, and wherein the indicator reagent zone includes a reagent capable, directly or indirectly, of immobilising an enzyme-labelled reagent in an amount dependent upon the assay result, characterised in that the developing solution includes a signal producing substrate for the enzyme. The substrate moves slower through the absorbent material than the enzyme-labelled reagent or any compound of the enzyme-labelled reagent formed in the assay. The absorbent material is suitably in the form of an elongate strip provided with transverse reagent zones. The device is useful for performing immunoassays including immunometric assays and dual analyte assays.

This application is a continuation of application Ser. No. 08/083,329,filed Jun. 29, 1993, now abandoned, which is a continuation ofapplication Ser. No. 07/702,128, filed May 16, 1991, now abandoned,which is a continuation of application Ser. No. 07/347,725, filed May 5,1989, now abandoned, which is a continuation of application Ser. No.07/080,565, filed Jun. 29, 1987, now abandoned.

FIELD OF THE INVENTION

This invention relates to a device for performing an enzyme-labelledbinding assay, the device comprising an absorbent material and adeveloping solution, wherein the absorbent material is provided with aplurality of reagent zones including an indicator reagent zone and iscapable of transporting the developing solution by capillary actionsequentially through each reagent zone, and wherein the indicatorreagent zone includes a reagent capable, directly or indirectly, ofimmobilising an enzyme-labelled reagent in an amount dependent upon theassay result.

BACKGROUND TO THE INVENTION

Binding assays such as immunoassays, are in widespread use in clinicallaboratories for the detection of substances in biological fluids. Thereis however increasing interest in the development of assays which can beperformed without the need for complex analytical techniques andequipment, for example, by a physician in his consulting room or by apatient at home. Such assays are not only more convenient but allowsavings in time and expense. Particular applications for whichconvenient and simple assays and reagent formulations are being soughtare the detection of pregnancy and of the fertile period of themenstrual cycle.

It is known to conduct binding assays on a strip of material providedwith a plurality of reagent zones, in which a developing solution formsa solvent front which passes along the strip by capillary action pickingup and facilitating reaction between a sample and assay reagents locatedat the reagent zones (see for example, British patent specificationGB-B-1589234). A feature of such strips is the existence of a testlocation at which, under certain conditions determined by the assayprotocol and the sample composition, a labelled reagent becomesimmobilised, giving an indication of the assay result. In early assays,the labelled reagent was a binding partner or analogue of the analyte tobe measured, labelled with a radioactive isotope. Such assays requireinstrumentation to detect the level of radioactive label and may presenthealth risk problems. A solution to this has been the use of enzymelabels which produce a characteristic signal (such as a colorimetricsignal) with an appropriate substrate.

A significant problem in the design of such so called "dipstick"enzyme-labelled binding assays is the application of the appropriateenzyme substrate in order to produce a detectable signal. The signal maybe developed by adding substrate to the appropriate position on thereagent strip after allowing the assay to proceed to completion.Alternatively, the appropriate part of the strip may be removed andchemically analysed. All of these represent steps which would be atleast inconvenient, if not impossible for home use of the assay.

SUMMARY OF THE INVENTION

According to the present invention, we provide a device for performingan enzyme-labelled binding assay, the device comprising an absorbentmaterial and a developing solution, wherein the absorbent material isprovided with a plurality of reagent zones including an indicatorreagent zone, and is capable of transporting the developing solution bycapillary action sequentially through the reagent zones, and wherein theindicator reagent zone includes a reagent capable, directly orindirectly, of immobilising an enzyme-labelled reagent in an amountdependent upon the assay result, characterised in that the developingsolution includes a signal-producing substrate for the enzyme.

The present invention, in its broadest concept, facilitates the use ofbinding assays in the home with the minimum of manipulative steps. Asthe solvent front of the developing solution passes through theabsorbent material, picking up the reagents and allowing them to react,a signal, such as colour formation, occurs when the substrate contactsthe enzyme-labelled reagent. This may result in a band of signal at thesolvent front of the developing solution which passes up the test stripin the course of the assay. As the solvent front passes through areagent zone in which the enzyme-labelled reagent is immobilised, asignal separation occurs as the bound enzyme-label is immobilised andunbound enzyme-label proceeds with the solvent front.

This separation, whilst adequate for many applications, may beunsatisfactory because the separation of the signal resulting from boundand unbound enzyme-labelled species is, under some binding conditionsunclear. In a colorimetric assay this may result in smearing of thecolour signal and obscurity in the assay result.

Preferably, in a particularly advantageous form of the device, thedeveloping solution includes a signal-producing substrate for the enzymewhich substrate, in use, is transported by the developing solutionslower than the enzyme-labelled reagent or any compound of theenzyme-labelled reagent formed in the assay.

The preferred form of the invention overcomes the further problemdescribed above because transport of the enzyme substrate occurs slowerthan transport of the enzyme-labelled reagent. In use, the reactionsinvolved in the assay take place in the moving solvent front as thedeveloping solution passes through the absorbent material. The substrateis transported slower than the enzyme-labelled reagent and compoundsformed during the assay reactions, which remain ahead of the substratein the absorbent material. The assay result is given by immobilising theenzyme-labelled reagent, either directly or indirectly through anotherspecies. The enzyme-labelled reagent, thus immobilised, does not remainahead of the substrate which subsequently comes into contact with anyimmobilised enzyme-labelled reagent thus generating a signal. Anyenzyme-labelled reagent which is not immobilised remains ahead of thesubstrate and therefore colour smearing does not occur. In the absenceof immobilised enzyme-labelled reagent, no signal is generated in theimmobilising region of the absorbent material at any stage in the assay,not even transiently as the solvent front passes through theimmobilising region. This is important, especially when the device isintended for home use, to show a clear result. The invention provides asignificant improvement to the sensitivity and clarity of dip stickassays.

The signal-producing substrate may be a single colour-producing compoundor may be a compound acting as a cofactor with a further compound orcompounds to produce a coloured signal in the presence of enzyme. Thefurther compound or compounds may be included in the developing solutionand may either be transported by the developing solution with, or moreslowly than, the enzyme-labelled reagent or any compound of theenzyme-labelled reagent formed in the assay. For example, the substratemay be tetramethylbenzidine which is oxidised by hydrogen peroxide inthe presence of a peroxidase to produce a coloured signal.

The assay may be any type of enzyme-labelled binding assay in which theamount of an enzyme-label immobilised in an indicator reagent zone isindicative of the result of the assay. The signal produced in theindicator reagent zone is preferably colorimetric. The device issuitable for conducting competitive and non-competitive binding assaysin which analyte in the sample either binds to an enzyme-labelledreagent or binds to an enzyme-labelled reagent in competition with ananalyte analogue. Preferably the assay is an immunoassay.

The assay may be for example a two site immunometric assay or a dualcompetition assay such as a dual analyte assay of the type described inpublished British specifications GB-B-2029011 and GB-B-2116313. Theanalyte may be any analyte which has a specific binding partner.

The absorbent material, which may be in the form of an elongate strip,may be any material capable of transporting the developing solution bycapillary action. A preferred material is a bibulous paper, such as aglass fibre paper, although any material exhibiting the necessarycapillary property and a low level of non-specific binding could beused.

Differential migration of the enzyme-labelled species and the substratemay be achieved by selection of appropriate materials for the absorbentmaterial given particular combinations of enzyme-label, substrates andbuffers. Compounds may be added to the paper to modify the differentialtransport properties of the paper. In particular, a compound orcompounds may be added to the paper to increase attractive interactionsbetween the paper and the substrate relative to interactions between thepaper and the enzyme-labelled species. For example, where the substrateis tetramethylbenzidine (TMB) and the paper is borosilicate glass fibrepaper, an acrylic binder incorporated in the glass fibre duringmanufacture reduces the migration rate of TMB markedly. Converselycertain compounds such as B-cyclodextrin interfere with the interactionand reduce the attractive effect of such binders. We have demonstratedthat, on a strip of glass fibre composed of pure borosilicate glass,peroxidase, E₂ O₂ and TMB migrate with the solvent front (Rf=1.0) whenan aqueous developing buffer such as 0.1M acetate (pH 6.0) containing0.2% Tween 20 is employed. However, when an acrylic binder is includedin the manufacture of the borosilicate glass fibre, the migration of H₂O₂ and peroxidase is unaltered but the migration rate of TMB is reduced(Rf less than 1.0). The Rf value of the TMB can thus be controlled tothe desired value by the concentration of acrylic binder present in theglass-fibre. The differential migration of TMB on glass-fibre containingan acrylic binder can also be controlled by the use of a compound whichinterferes with the interaction between the TMB and the acrylic binder,e.g. B-cyclodextrin. For example, if a strip of Gelman AP25 extra thickglass-fibre paper (Gelman Sciences Inc., Ann Arbour, Mich., USA) isplaced into a solution containing 0.1M acetate (pH 6.0), 0.1 mgm1⁻¹ TMB,0.001% H₂ O₂ 1% DMSO and 0.2% Tween 20, the TMB migrates with an Rfvalue of 0.4. Inclusion of B-cyclodextrin in the developing solution at0.1% (w/v) increases the Rf value of TMB to 0.55 with no effect on theH₂ O₂. Increasing the B-cyclodextrin concentration to 0.25% (w/v)increases the Rf value of TMB to 0.7 with no effect on the H₂ O₂ . Inaddition, B-cyclodextrin has no effect on the migration of peroxidasewhich migrates with the buffer front (Rf=1.0).

Alternatively, differential migration of the enzyme-labelled species andthe substrate may be achieved by providing a substrate binding reagentzone, capable of binding the substrate at a location on the assay deviceencountered, in use, by the developing solution prior to theenzyme-labelled reagent zone, such that, in use, the substrate isprevented from passing through the said binding reagent zone until thebinding reagent zone is substantially saturated.

The reagent zones on or in the absorbent material are arranged such thatthe developing solution contacts them sequentially. The reagent zonesinclude the reagents for the particular assay protocol and may bearranged on or in the material to allow for a predetermined incubationperiod between contact with adjacent reagent zones.

The spacing of the reagent zones provides a parameter which may bevaried to set the predetermined incubation period. Alternatively themigration rate of the developing solution may be controlled or modifiedby the inclusion of a compound, such as a polymer, into the developingsolution. Suitable such polymers include dextran or polyvinylpyrrolidinewhich cause a reduction in the migration rate. The compound capable ofmodifying the migration rate of the developing solution (for example apolymer) may be provided in a further reagent zone immediately followinga given reagent zone such that, in use, the migration of reagents in thesolvent front will be temporarily halted or delayed at the given reagentzone whilst the compound at the further reagent zone is solubilised.Thus, by halting migration of the developing solution at a given reagentzone, more time is provided for incubation at that zone.

In the preferred form of the device, the absorbent material is in theform of an elongate strip with transverse reagent zones.

Preferably the cross-sectional area of the absorbent material in atleast part of the indicator zone is smaller than the cross-sectionalarea of the absorbent material in the rest of the device. Where theabsorbent material is in the form of a strip this may advantageously beachieved by forming a neck in the strip of the indicator reagent zone.This provides the additional advantage of concentrating all the reagentspassing up the strip of the reagent zone, thus increasing the potentialsignal intensity.

The developing solution may be the sample itself to which substrate isadded, but is preferably separate from the sample. Advantageously it iscontained in a rupturable sac adjacent part of the absorbent material,suitably at one end of a strip of absorbent material. In thealternative, the device of the invention may be in the form of a kitcomprising separately an absorbent material and a container of thedeveloping solution, as defined. Preferably, the developing solutioncomprises a buffer compatible with the assay system. A particularlypreferred developing solution for an enzyme-labelled immunoassaycomprises 0.1M acetate (pH 6) containing 0.2% (v/v) Tween 20 and theenzyme substrate, as appropriate. The developing solution may, inaddition, include a compound capable of modifying the migration rate ofthe developing solution, such as a polymer, for example dextran orpolyvinylpyrrolidone.

The enzyme may be any enzyme capable of producing a measurable signal inthe presence of an appropriate substrate. For example the enzyme may behorseradish peroxidase and the developing solution may containtetramethylbenzidine (TMB) and hydrogen peroxide.

The device may include a sample receiving zone which can, if desired, beprovided with a filter member, such as a filtration pad, to remove solidmaterial such as cellular material and debris.

In order that the physical dimensions of the absorbent material are notexcessive, the enzyme-labelled reagent (in its free and bound states)should preferably exhibit an R_(f) value of not less than 0.7.Preferably the R_(f) value of the enzyme substrate should be in therange 50 to 90% that of the enzyme-labelled species (in its free andbound states).

The device of the invention may include an assay completion indicatorzone comprising immobilised enzyme to indicate completion of the assay.Where the absorbent material is in the form of a strip, the assaycompletion indicator zone is preferably located near the end of thestrip remote from the end at which the developing solution is applied.

The absorbent material may be enclosed within a non-transparent coveringexcept in the indicator reagent zone where a transparent window may beprovided. Access to the sample zone may be provided by removal of aresealable plug which can be replaced after application of the sample.Application of a sample to the device may be by way of an applicatorwhich delivers a predetermined volume of the sample, for example asampling loop.

The device may be individually packaged, but for easy monitoring of themenstrual cycle, for example for home use, a plurality of devices of theinvention may be packaged together. We further provide therefore a testsheet comprising a plurality of devices of the invention.

In use, the developing solution is applied to the absorbent material.For example, where the absorbent material is in the form of a strip, thedeveloping solution is applied to one end of the strip, advantageouslyby rupturing a sealed sac, for example, by finger pressure, to releasethe contents. The developing solution advances through the absorbentmaterial, picking up sample applied at the sample receiving zone, andother reagents including an enzyme-labelled reagent. The enzymesubstrate or a cofactor included in the developing solution, travelsthrough the absorbent material more slowly than the enzyme-labelledreagent and no signal is therefore produced. The assay reactions takeplace in the advancing solvent front of the developing solution andafter an incubation period determined by the separation of the reagentzones, an amount of the enzyme-labelled reagent is immobilised in theindicator reagent zone in an amount dependent upon the assay result. Thesubstrate then comes into contact with the immobilised enzyme-labelledreagent, thereby generating a signal in the indicator reagent zone.

The result of an assay as indicated by the device of the invention maybe qualitative, read simply by the absence or presence of a signal,especially a coloured signal at the indicator reagent zone. This type ofresult may be, for example, of considerable use where a threshold valueof a particular analyte in a sample is being monitored (such as thelevel of a particular hormone). However, the device can be employed toprovide quantitive assay results. The intensity of the signal producedat the indicator reagent zone will be either proportional to orinversely proportional to the concentration of analyte present in thesample. Thus, the indicator reagent zone of the device may, following anassay, be inserted into a reflectance spectrophotometer, or afluorimeter (if the signal produced is fluorescent), to measure theintensity of the signal produced. Alternatively, the indicator reagentzone may be elongated in the direction of developing solution migrationor a plurality of individual indicator reagent zones may be provided.Thus, the length of signal produced at the indicator reagent zone or thenumber of individual zones which exhibit the signal will be quantitativeand proportional to, or inversely proportional to, the concentration ofanalyte present in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is now described by way of example with reference to theaccompanying drawings in which:

FIG. 1 shows a device for conducting a competitive hapten assay,

FIG. 2 shows a device for conducting a non-competitive hapten assay,

FIG. 3 shows a device for conducting a two-site sandwich, orimmunometric assay, and

FIG. 4 shows a device for conducting a dual analyte assay,

FIG. 5 shows a further device for conducting a dual analyte assay,

FIG. 6 shows test sheet comprising a plurality of devices of theinvention arranged to monitor the menstrual cycle,

FIG. 7 shows a device for conducting a competitive haptan assay,

FIG. 8 shows a device for conducting a non-competitive hapten assay, and

FIG. 9 shows a device for conducting a dual analyte assay.

Embodiments of the invention are described first generally withreference to FIGS. 1 and 6 and then more specifically with reference tospecific Examples 1 to 4.

The following description of materials and methods applies to thegeneral embodiments described below and specifically to Examples 1 and 4below.

Unless otherwise stated, all reagents were obtained from Sigma ChemicalCompany, Poole, U.K.

ABSORBENT STRIP MATERIAL

Gelman AP25 extra thick glass-fibre paper (from Gelman Sciences Inc.,Ann Arbor, Mich., U.S.A.)

DEVELOPING SOLUTION (SUBSTRATE BUFFER)

The developing solution was prepared as follows: To 1000 ml of sterileDH₂ O was added:

    ______________________________________                                        2.5          g B-cyclodextrin                                                 +8.2         g sodium acetate                                                 +0.357       g citric acid                                                    +50          ul 30% H.sub.2 O.sub.2                                           +10          ml of TMB in DMSO at 10 mg/ml                                    +5           g of BSA *                                                       +1           ml Tween 20                                                      +5           g sodium chloride                                                ______________________________________                                         * Sigma Chemical Company No.A3647, Fraction V                            

SOLID-PHASE ANTIBODY

Reagent immobilisation on the strip, may be accomplished by physicaladsorption or chemical coupling to the strip using techniques well knownin the art (see R. Axen et al, (1967), Nature, 214, 1302; S, Avrameasand T. Ternynck, (1969), Immunochemistry, 6, 53: G. S. Bethell et al,(1979), The Journal of Biological Chemistry, 254, 2672; J. M. J.Frechet, (1981), Tetrahedron, 37, 663).

A preferred technique is however to attach the ligand to an insolubleparticle which is of the correct size to be trapped within the frameworkof the absorbent strip material and thus unable to move with thedeveloping solution. A suitable type of particle is Eupergit ClZ,supplied by Rohm Pharma GmbH, Weiterstadt, West Germany. The coupling ofantibodies to this material is described below:

Buffer A.

To 1000 ml of DH₂ O was added:

    ______________________________________                                                     5.96 g Na.sub.2 HPO.sub.4                                                    +1.24 g NaH.sub.2 PO.sub.4                                                   +29.22 g NaCl                                                      ______________________________________                                    

To 500 ul of the above buffer (A) was added 3-4 mg of freeze-driedantibody and 125 mg of Eupergit ClZ. The reagents were mixed briefly andthen left to stand at room temperature for 48 hours. The Eupergit wasthen resuspended in 20 ml of the following buffer (Buffer B).

Buffer B.

To 1000 ml of DH₂ O was added:

    ______________________________________                                                    5.96 g Na.sub.2 HPO.sub.4                                                    +1.24 g NaH.sub.2 PO.sub.4                                                    +3.75 g Glycine                                                    ______________________________________                                    

The Eupergit ClZ was allowed to settle at 4° C. for 12 hours. Thesupernatant was then aspirated off, and the Eupergit then resuspended in20 ml of Buffer B and again allowed to settle. The supernatant was thenagain aspirated off, and the Eupergit/antibody resuspended in 12.5 ml ofBuffer B.

ANTIBODY-PEROXIDASE CONJUGATE

The peroxidase conjugate may be prepared using, for example,sulfhydrylmaleimide coupling (Ishikawa, E., (1980), Immunoassay suppl,1, 1-16; Duncan, R. J. S. et al, Anal. Biochem., 132, 68-72),disulphide-thiol exchange (Carlsson, J., et al, (1978), Biochem, J.,173, 723-737), periodate oxidation (Nakane, P. K., et al (1974), J.Histochem. Cytochem, 22, 1084-191) or glutaraldehyde coupling (Avrameas,S. (1969), Immunochem., 6, 43-72; Avrameas, S., et al, (1971),Immunochem, 8, 1175-1179).

In the preferred method, horseradish peroxidase (HRP) was conjugated toa monoclonal antibody using an adaptation of the glutaraldehyde methodof Avrameas (loc. cit.). 100 mg of HRP was dissolved in 500 ul of 0.05Mbicarbonate buffer (pH 9.5) to which was added 500 ul of 11% (w/v)glutaraldehyde prepared in the same buffer. The reaction was conductedat room temperature (20° C. -25° C.) for two hours with gentle shaking.The reaction mixture was then applied to a PD10 column (Pharmacia Ltd.)which had previously been equilibrated with 0.05M bicarbonate buffer (pH9.5). Elution was achieved with the same buffer and those fractionscontaining activated HRP were pooled. Antibody (2-3 mg/ml) in 0.05Mbicarbonate buffer (pH 9.5) was added to the activated HRP to give amass ratio of 6:1 of activated HRP to antibody. The reaction wasconducted at 4° C. for 16-21 hours after which the antibody-HRPconjugate was purified by gel filtration, typically on a TSK G3000SWcolumn (Toya Soda, Japan).

MIXED STEROID ANTIGEN (MSA)

Mixed steroid antigen (MSA) is a bifunctional ligand comprisingoestrone-3-glucuronide (E13G) and pregnanediol-3-glucuronide (PD3G). Thesynthesis of the compound is described in British patent specificationGB-B-2116318.

Buffer C.

To 1000 ml of sterile DH₂ O was added:

8.2 g sodium acetate

0.357 g citric acid

2 ml Tween 20

FIG. 1 shows a device for measuring the concentration ofpregnanediol-3-glucuronide (PD3G) in a urine sample using a competitivehapten assay protocol. Referring to FIG. 1, the device comprises astrip, 1, of a bibulous paper and a reservoir, 2, containing adeveloping solution, 3, consisting of a substrate-buffer. The strip isprovided with a sample receiving zone, 4, a first reagent zone, 5, and asecond, indicator, reagent zone, 6. The first reagent zone, 5, includesenzyme-labelled PD3G hapten covalently attached to a horseradishperoxidase enzyme-label, the enzyme-labelled PD3G being impregnated intothe strip, such that, in use, it is caused to migrate through the stripby passage of the developing solution through the strip. The indicatorzone, 6, comprises antibody to PD3G covalently bonded to the strip. Thereservoir, 2, consists of a rupturable sac containing the substratebuffer which includes the cofactors tetramethylbenzidine and hydrogenperoxide. In use, a sample of urine is applied to the sample receivingzone, 4, and the substrate buffer, 3, is released onto the end of thestrip by rupturing the sac, 2, with finger pressure. The developingsolution, 3, passes through the strip by capillary action picking up thesample and the enzyme-labelled PD3G in the solvent front from the samplereceiving zone, 4, and the first reagent zone, 5, respectively. Theenzyme cofactor, tetramethylbenzidine, is carried through the strip moreslowly than the enzyme-labelled PD3G. Thus, when the solvent front ofthe substrate buffer, 3, reaches the first reagent zone, 5, little or nocofactor is present in the solvent front and substantially no colourformation occurs. Any PD3G present in the sample competes with theenzyme-labelled PD3G for a limited number of binding sites in thesecond, indicator, reagent zone, 6. The amount of enzyme-labelled PD3Gthat becomes bound to the strip in the indicator reagent zone, 6, istherefore inversely proportional to the concentration of PD3G in thesample. Continued development of the strip washes any unreacted reagentsthrough the indicator zone, 6, and subsequently brings the cofactor intocontact with the indicator zone where the substrate is converted by thebound enzyme-labelled PD3G to give a coloured product (horseradishperoxidase catalyses the oxidation of tetramethylbenzidine by hydrogenperoxide). The coloured product does not migrate further, thus giving asharp band of colour at the indicator zone, 6. The difference betweendistances B and A as shown in FIG. 1 is made relatively small as thesample and the enzyme-labelled PD3G do not interact until the indicatorzone, 6, is reached thus allowing a reduction in the length of thestrip. However, the distance B must be sufficient to permit adequatewashing of the bound enzyme-labelled PD3G by continued migration of thedeveloping solution, 3, prior to the arrival of the cofactor at theindicator zone, 6. The duration of the competitive reaction which occursat the indicator zone, 6, is equal to the time it takes for the sampleand the enzyme-labelled PD3G to pass through the indicator zone, 6. Thisis determined by the physical properties of the material from which thestrip is made.

FIG. 2 shows a further device for measuring the concentration of PD3G ina urine sample using a non-competitive hapten assay protocol. Referringto FIG. 2, the device comprises a strip, 11, of a bibulous paper and areservoir, 12, containing a developing solution, 13 consisting of asubstrate-buffer. The strip is provided with a sample receiving zone,14, a first reagent zone, 15, and a second, indicator, reagent zone, 16.The first reagent zone, 15, includes enzyme-labelled anti-PD3Gcomprising antibody to PD3G covalently attached to a horseradishperoxidase enzyme-label, the enzyme-labelled anti-PD3G being impregnatedinto the strip such that, in use, it is caused to migrate through thestrip by passage of the substrate-buffer, 13, through the strip. Theindicator zone, 16, comprises PD3G covalently bonded to the strip. Thereservoir, 12, is as described above with reference to FIG. 1.

In use, a sample of urine is applied to the sample receiving zone, 14,and the substrate-buffer, 13, is released onto the end of the strip byrupturing the sac, 12, with finger pressure. The substrate buffer, 13,passes through the strip by capillary action picking up the sample. AnyPD3G present in the sample is bound in the first reagent zone, 15, bythe enzyme-labelled anti-PD3G which is present in excess. The incubationtime for this interaction to take place is controlled by the differencebetween distances B and A as shown in FIG. 2. As the solvent frontcontacts the indicator zone, 16, PD3G covalently bonded to the stripbinds any unreacted enzyme-labelled antibody to PD3G. Again, since thecofactor travels more slowly through the strip than the enzyme-labelledantibody, colour generation only occurs at the indicator zone, 16, wherethe enzyme-labelled antibody has been immobilised.

FIG. 3 shows a device for measuring the concentration of thyroidstimulating hormone (TSH) in a sample using a two-site sandwich assay orimmunometric assay protocol. Referring to FIG. 3 the device comprises astrip 21 of a bibulous paper and a reservoir, 22, containing adeveloping solution, 23 consisting of a substrate-buffer. The strip isprovided with a sample receiving zone, 24, a first reagent zone, 25, anda second, indicator, reagent zone, 26. The first reagent zone, 25,includes enzyme-labelled anti-TSH comprising antibody to TSH covalentlyattached to a horseradish peroxidase enzyme-label, the enzyme-labelledanti-TSH being impregnated into the strip, 21, such that, in use, it iscaused to migrate through the strip by passage of the substrate-buffer,23. The indicator zone, 26, comprises a second antibody to TSHcovalently bonded to the strip, 21. The second antibody has specificityfor a different and non-competing epitope of TSH from that of theenzyme-labelled antibody. In use, any TSH present in the sample is boundby the enzyme-labelled antibody which is present in excess as theyco-migrate through the strip. The time allowed for this first reactionis governed by the difference between the distances B and A as shown inFIG. 3. On passing through the indicator zone, the TSH (in the form ofan antibody complex) is bound by the second antibody. The time of thesecond incubation is governed by the speed of capillary migration of thesolvent front through the material of the strip. Again, colourdevelopment only occurs where the enzyme-labelled antibody isimmobilised in the indicator zone thus allowing the cofactor to bebrought into contact with it by the capillary motion of the solventfront.

FIG. 4 shows a device for measuring the ratio of the concentrations ofPD3G and oestrone-3-glucuronide (E13G) in a urine sample. The ratio ofthese two products has been shown to be indicative of the fertile periodof the female menstrual cycle (see for example British publishedspecifications GB-B-2029011 and GB-B-2116318).

Referring to FIG. 4, the device comprises a strip, 31, of bibulous paperand a reservoir, 32, containing a developing solution, 33 consisting ofa substrate-buffer. The strip is provided with a sample receiving zone,34, a first reagent zone, 35, a second reagent zone, 36, a third reagentzone, 37, and a fourth, indicator, reagent zone, 38. The first reagentzone, 35, comprises a mixed steroid antigen (MSA) consisting of a PD3Ghapten and an E13G hapten covalently bonded to a bridging structure. TheMSA is impregnated into the strip, 31, such that, in use, it may migratethrough the strip in the advancing solvent front of thesubstrate-buffer, 33. The second reagent zone, 36, comprises an antibodyto E13G which may be free to migrate through the strip in the advancingsolvent front though is preferably covalently bonded to the strip. Thethird reagent zone, 37, comprises an enzyme-labelled anti-E13G (antibodyto E13G covalently attached to a horseradish peroxidase enzyme-label),impregnated into the strip, 31, such that it may migrate through thestrip with the substrate-buffer, 33. The fourth, indicator, reagentzone, 38, comprises antibody to PD3G covalently bonded to the strip. Thereservoir, 32, comprises a rupturable sac containing thesubstrate-buffer.

In use, a urine sample is applied to the sample zone, 34, and thereservoir, 32, of developing solution, 33, is ruptured, releasing thesubstrate-buffer onto one end of the strip. The substrate-buffer, 33,passes up the strip, 31, by capillary action picking up sample from thesample receiving zone, 34, and mixed steroid antigen from the firstreagent zone, 35. The sample and the MSA co-migrate along the strip tothe second reagent zone, 36, at which the antibody to E13G is covalentlyimmobilised. The MSA and E13G present in the sample compete for limitedbinding sites as they pass through the second reagent zone, 36. If theconcentration of E13G in the sample is low, then a substantialproportion of the MSA is bound in the second reagent zone, 36, andcannot migrate further. If, however, the sample concentration of E13G ishigh, then the MSA will be free to migrate, together with the sample, tothe next reagent zone, namely, the third reagent zone, 37, comprisingenzyme-labelled antibody to E13G. This latter reagent is present inexcess and is non-covalently absorbed to the strip, 31. Theenzyme-labelled antibody binds to the MSA as they both migrate togetheralong the strip to the fourth, indicator, reagent zone, 38, at whichantibody to PD3G is covalently attached to the strip. At the fourthindicator reagent zone, 38, any PD3G present in the sample competes withthe MSA/anti-E13G complex for binding to a limited number of bindingsites of the covalently immobilised anti-PD3G antibody. Theenzyme-labelled immunocomplex will be bound at the indicator zone, 38,only when the sample concentration of PD3G is low. The excess reagentsare washed from the measuring location by continued development of thestrip. On reaching the indicator zone, 38, the cofactor is converted bythe enzyme-labelled, antibody-bound MSA to a coloured product giving aclear positive signal. The assay can be tuned to give a positiveresponse only when a predetermined elevated level of E13G coincides witha predetermined elevated level of E13G. A further reagent zone (notshown) may be provided at a point remote from the reservoir, 32, whichcomprises covalently bound horseradish peroxidase. This reagent zonegives an indication that substrate has migrated through the length ofthe strip thus indicating that the assay has run to completion.

FIG. 5 shows a further device for measuring the ratio of theconcentrations of PD3G and E13G in a urine sample.

Referring to FIG. 5, the device comprises a strip, 41, of bibulous paperand a reservoir, 42, containing a developing solution. The strip isprovided with a sample receiving zone, 43, a first reagent zone, 44, asecond reagent zone, 45, a third reagent zone, 46, a fourth reagentzone, 47, a fifth reagent zone, 48, a sixth, test indicator, reagentzone, 49 and an optional seventh, control indicator, reagent zone, 50.The first reagent zone, 44, comprises anti-E13G (antibody to E13G). Thesecond reagent zone, 45, comprises a mixed steroid antigen (MSA) asdescribed in Example 4. The third reagent zone, 46, comprisesbiotin-labelled anti-PD3G dextran-coated charcoal. The fifth reagentzone, (antibody to PD3G covalently bonded to biotin). The fourth reagentzone, 47, comprises 48, comprises enzyme-labelled anti-E13G (antibody toE13G covalently bound to horseradish peroxidase). The sixth reagentzone, 49, comprises immobilised streptavidin. The optional seventhreagent zone, 50, comprises horseradish peroxidase.

The active components of the reagent zones are dried onto the strip astransverse bands. The streptavidin in the sixth, test indicator, reagentzone, 49, is covalently attached to the strip and the dextran-coatedcharcoal in the fourth reagent zone, 47, is deposited onto the strip byadding an aqueous suspension of microparticulate charcoal andsubsequently removing the water. All the other reagents are soluble andare impregnated into the strip by applying them each in solution andsubsequently drying. The soluble reagents, in use, migrate with thesolvent front of the developing solution. The reservoir, 42, comprises arupturable sac containing the developing solution.

In use, a urine sample is applied to the sample receiving zone, 43, andthe reservoir 42, of developing solution is ruptured, releasing thedeveloping solution onto one end of the strip. The developing solutionpasses up the strip by capillary action, picking up sample from thesample receiving zone, 43, and, in sequence, anti-E13G, MSA andbiotin-labelled anti-PD3G. The soluble components of the assay passthrough the strip in the advancing solvent front, and at the same timeare mixed and allowed to react. The separation of the reagent zones maybe adjusted to facilitate optimum incubation times for reaction.Unbound, low molecular weight species such as MSA and steroids areremoved from the solvent front by the charcoalin the fourth reagentzone, 47. The solvent front passes through the fifth reagent zone, 48,picking up enzyme-labelled anti-E13G, thus completing the assayprotocol. The presence of complexes of enzyme-labelledanti-E13G/MSA/biotin-labelled PD3G is indicative of a high level of E13Gand a low level of PD3G. Such complexes are immobilised in the sixth,test indicator, reagent zone, 49, by the interaction of biotin withimmobilised streptavidin.

The developing liquid, as previously stated, includes a colour producingsubstrate for peroxidase. If the substrate has an R_(f) valuesubstantially the same as the enzyme-labelled species in the device,colour will develop in the solvent front as soon as the fifth reagentzone, 48, is reached and the solvent front will remain coloured for therest of its passage through the strip. The presence of colour at the endof the strip remote from the developing solution indicates completion ofthe assay. As the solvent front meets and passes through the sixth, testindicator, reagent zone, 49, any coloured products and unboundenzyme-labelled anti-E13G will be washed clear of the zone by incomingfresh developing solution. However, if the complex includingenzyme-labelled anti-E13G has become immobilised, colour generationoccurs in the sixth, test indicator, reagent zone. Thus colour in thesixth reagent zone indicates a positive test result. Alternatively, ifthe substrate has an R_(f) value less than the R_(f) value of theslowest moving enzyme-labelled species, colour generation will onlyoccur where an enzyme-labelled species is immobilised, i.e. in the sixthreagent zone when a positive result is obtained. In this alternative,which is preferred, a seventh, control indicator, reagent zone, 50,comprising horseradish corresponding to completion of the assay.peroxidase may be provided to indicate arrival of the substrate at apredetermined part of the strip,

FIG. 6 shows a test sheet for monitoring the menstrual cycle embodying aplurality of test strips as described above with respect to FIGS. 4 or5.

Referring to FIG. 6, the test sheet comprises a rigid plastics backingplate or stand, 51, supporting a plurality of test strips of theinvention, e.g. 52 (In the Figure the details of the absorbent stripsare not shown). In the embodiment shown, fifteen test strips areprovided in side-by-side parallel arrangement. The backing plate orstand, 51, is overlaid with a plastics film, to cover the test strips,apart from in the sample receiving zone, 53. The plastics film (shown inFIG. 6 as transparent for clarity) is opaque save in the test indicatorzones, 54, and in the control indicator zones, 55. The plastics film maybe suitably masked or printed to indicate clearly the sample receiving,test indicator and control indicator zones. The developing solution iscontained in separate rupturable sacs, 56, one for each test strip.

In use, mid-stream urine is sampled using a disposable sample loop andan aliquot is blotted onto the sample receiving zone, 53, of a teststrip, 52. The seal of a rupturable sac, 56, of developing solution isbroken by finger pressure, thus initiating the test. After 15 to 20minutes, the control indicator zone, 55, is observed and, if coloured,the assay result is read from the test indicator zone, 54.

The presence of colour in the test zone indicates a positive result i.e.the woman is in, or near, her fertile period. The converse applies withthe absence of colour. For the woman who wishes to avoid conception, itis intended that she should test her urine once per day starting atabout day 6 or 7 of her cycle. She should continue daily testing until aperiod of sustained positive results have been observed (more than 2days) followed by a period of sustained negative results (more than 2days). This would normally mean a total of 10 to 15 tests in a typicalcycle. Whenever a positive result is observed, the woman should refrainfrom intercourse and should continue to do so until two successive dailynegative results have been observed.

It is intended that colours developed in the strips are stable, and soform a semi-permanent record of the woman's cyclical activity.

EXAMPLE 1

A device for conducting a competitive hapten assay similar to thatdescribed generally with respect to FIG. 1 was prepared.

Referring to FIG. 7, the device comprises a strip of Gelman glass-fibre(15×1 cm), 61, and a reservoir, 62, containing the substrate-buffer, 63.The strip is provided with a sample receiving zone, 64, (1.5 cm from thelower end of the strip), a first reagent zone, 65, (2.0 cm from thelower end of the strip) to which has been applied 10 μl MSA (1000 nM inBuffer C), a second reagent zone, 66 (2.5 cm from the lower end of thestrip) to which has been applied 10 μl of a monoclonal antibody to PD3Gconjugated to peroxidase (1 μg ml⁻¹ in Buffer C) and an indicator zone,67, (5.0 cm from the lower end of the strip) to which has been applied50 μl of a solid phase monoclonal antibody to E13G.

In use, 20 μl of sample to be assayed for PD3G is applied to the samplereceiving zone, 64, and the the strip is immersed. After 15 min, theindicator lower end of the strip is placed into 2 ml of substrate-buffersuch that only the first 0.5 cm of zone, 67, is observed. The presenceof a blue colour at the indicator zone, 67, shows that the concentrationof PD3G in the sample is less than 1000 nM. (colour at 5000 and 10000nM: no colour at 15000, 20000 and 25000 nM)

The device operates as follows:

As the substrate-buffer migrates along the strip, PD3G in the sample,MSA and anti-PD3G peroxidase conjugate are transported at the bufferfront together with all the components of the buffer-substrate exceptTMB which exhibits a slower rate of migration (Rf=0.7). During migrationalong the strip, the PD3G and MSA compete for the binding sites of theanti-PD3G peroxidase conjugate. On reaching the indicator zone, 67, theimmobilised anti-E13G antibody binds to the MSA. If the concentration ofPD3G in the sample is low, then the MSA will also be bound by theanti-PD3G peroxidase conjugate and when the TMB reaches the indicatorzone, 67, a blue colour will be formed. If the concentration of PD3G inthe sample is high, then the anti-PD3G peroxidase conjugate will notbind the MSA and no colour signal will be observed at the indicatorzone, 67.

EXAMPLE 2

A device for conducting a non-competitive hapten assay similar to thatdescribed generally with respect to FIG. 2 was prepared.

Referring to FIG. 8 the device comprises a strip of Gelman glass-fibre(15×1 cm), 71, and a reservoir, 72, containing the substrate-buffer, 73.The strip is provided with a sample receiving zone, 74, (1.5 cm from thelower end of the strip), a first reagent zone, 75, (2.0 cm from thelower end of the strip) to which has been applied 10 ul of a monoclonalantibody to PD3G conjugated to peroxidase (1 ug ml⁻¹ in Buffer C), asecond reagent zone, 76, (5.5 cm from the lower end of the strip) towhich has been applied 10 ul of MSA (5,000 nM in Buffer C) and anindicator zone, 77, (6.0 cm from the lower end of the strip) to whichhas been applied 50 ul of a solid phase monoclonal antibody to E13G.

In use, 20 ul of sample is applied to the sample receiving zone, 74, andthe lower end of the strip is placed into 2 ml of substrate-buffer suchthat only the first 0.5 cm of the strip is immersed. After 15 min, theindicator zone, 77, is observed. The presence of a blue colour at thiszone, 77, means that the PD3G concentration in the sample is less than10000 nM. (colour at 5000 and 10000 nM: no colour at 15000, 20000 and25000 nM)

The device operates as follows:

As the substrate-buffer migrates along the strip, the PD3G in the sampleand the anti-PD3G peroxidase conjugate are transported at the bufferfront together with all the components of the buffer-substrate exceptTMB which exhibits a slower rate of migration (Rf=0.7). During migrationalong the strip, the anti-PD3G peroxidase binds the PD3G. On reachingthe second reagent zone, 76, the MSA in excess binds any unreactedanti-PD3G peroxidase conjugate, and is transported to the indicatorzone, 77, where the solid phase anti-E13G antibody binds both free andanti-PD3G peroxidase conjugate bound MSA. Thus, if the concentration ofPD3G present in the sample is low, most of the anti-PD3G peroxidaseconjugate will be bound by the MSA at the indicator zone, 77. When theTMB reaches the indicator zone, 77, a blue colour will be formed.However, if the concentration of PD3G in the sample is high, then mostof the anti-PD3G peroxidase conjugate will be unable to bind the MSA andthus no colour will be observed at the indicator zone, 77.

EXAMPLE 3

A device for conducting a two-site sandwich immunoassay for thyroidstimulating hormone (TSH) such as described generally with reference toFIG. 3 was prepared.

Referring again to FIG. 3, the strip, 21, is Gelman glass-fibre (17×1.8cm). At the first reagent zone, 25, (5.5 cm from the lower end of thestrip) is applied to 20 ul of a monoclonal antibody to TSH conjugated toperoxidase (5 ug ml⁻¹ in Buffer C). At the indicator zone, 26, 300 ul ofa solid-phase monoclonal antibody to TSH is applied (7.5 cm from thelower end of the strip). In use, 50 ul of sample is applied to thesample receiving zone, 24, and the strip is placed into 5 ml ofsubstrate-buffer such that only the first 1 cm of the strip is immersed.After 15 min, the indicator zone, 26, is observed. The presence of ablue colour indicates that the concentration of TSH present in thesample is greater than 200 mU/L: (No colour at 50, 100 and 200 mU/L:colour at 250 mU/L).

EXAMPLE 4

In another Example of a two-site sandwich assay of the type describedgenerally with reference to FIG. 2, the monoclonal antibodies to TSHdescribed in Example 3 above (at reagent zone 25 and indicator zone 26)were replaced with monoclonal antibodies to human chorionicgonadotrophin. The test can be used as a pregnancy indicator with colourbeing formed when the urine sample contains more than 200 mIU/ml hCG.(No colour at 50, 100 and 200 mIU/ml: colour at 250, 300 and 500mIU/ml).

EXAMPLE 5

A device for conducting a dual analyte assay similar to that describedgenerally with respect to FIG. 4 was prepared.

Referring to FIG. 9 the device comprises of a strip of Gelmanglass-fibre (15×1 cm), 81, and a reservoir, 82, containing thesubstrate-buffer, 83. The strip contains a sample receiving zone, 84,(1.5 cm from the lower end of the strip), a first reagent zone, 85, (2.0cm from the lower end of the strip) to which has been applied 10 ul of amonoclonal antibody to PD3G conjugated to peroxidase (1 ug ml⁻¹ inBuffer C), a third reagent zone, 87, (3.0 cm from the lower end of thestrip) to which is applied 10 ul of MSA (250 nM in Buffer C) and anindicator zone, 88, (5.0 cm from the lower end of the strip) to which isapplied 50 ul of a solid phase monoclonal antibody to E13G.

In use, 20 ul of a urine sample is applied to the sample receiving zone,84, and the strip is placed into 2 ml of substrate-buffer such that thelower 0.5 cm of the strip is immersed. After 15 min, the indicator zone,88, is observed. The presence of a blue colour at this zone, 88, meansthat the sample concentration of E13G is greater than 50 nM and that thesample concentration of PD3G is less than 10000 nM (see Table 1).

                  TABLE 1                                                         ______________________________________                                        PD3G (nM)                                                                     El3G (nM) 0       1000   5000   10000 20000                                   ______________________________________                                         0        -       -      -      -     -                                        50       -       -      -      -     -                                       100       +       +      -      -     -                                       200       +       +      -      -     -                                       ______________________________________                                         + = Blue colour at indicator zone.                                            - = No blue colour at indicator zone.                                    

The device operates as follows:

As the substrate-buffer migrates along the strip, the E13G and PD3G ofthe sample, anti-PD3G peroxidase conjugate, anti-E13G antibody and MSAmigrate at the buffer front with all the components of thebuffer-substrate except TMB which exhibits a lower rate of migration(Rf=0.7). If the sample concentration of E13G is low, then the MSA willbe bound by the anti-E13G antibody during the migration and will not bebound by the solid phase anti-E13G antibody at the indicator zone, 88.If the E13G level is high, then the MSA is free to bind at the indicatorzone, 88. If the PD3G concentration of the sample is low, then the MSAwill be bound by the anti-PD3G peroxidase conjugate and thus, when theTMB reaches the indicator zone, 88, a blue colour will be formed. If,however, the PD3G concentration of the sample is high, then it will bindto the anti-PD3G peroxidase conjugate preventing the latter from bindingto the MSA and thus no signal will be generated.

It will be understood that the invention has been described by way ofexample only and modifications of detail may be made within the scope ofthe invention.

We claim:
 1. A test system for performing a binding assay fordetermining the presence or absence of an analyte in a sample,comprising:a) an absorbent material in the form of an elongate striphaving a sample application zone, upstream of a plurality of transversereagent zones, wherein an enzyme-labelled reagent zone includes anenzyme-labelled species, comprising either an enzyme-labelled analyte oran enzyme-labelled reagent that binds to said analyte, such that in use,said enzyme-labelled species is caused to migrate through the strip bypassage of a developing solution through the strip, and wherein anindicator reagent zone includes an immobilized reagent that directly orindirectly binds, and thereby immobilizes, said enzyme-labelled speciesin an amount dependent on the quantity of said analyte present in saidsample; and b) a developing solution, wherein said developing solutioncomprises a signal-producing substrate for the enzyme which is a singlecolor-producing compound or a compound acting as a cofactor with afurther compound or compounds to produce a signal in the presence ofenzyme, wherein any of said further compound or compounds are present inthe developing solution, wherein said developing solution is initiallyin contact only with that portion of said absorbent material upstreamfrom said indicator zone but in which ultimately, by capillary action,sequentially contacts all reagent zones of said absorbent material, andwherein said signal-producing substrate is transported by the developingsolution slower than said enzyme-labelled species either by addition tosaid absorbent material at least one compound that increases theattractive interaction between said absorbent material and saidsubstrate relative to the attractive interaction between said absorbentmaterial and said enzyme-labelled species or by provision of a substratebinding reagent zone which binds said substrate at a location upstreamfrom said enzyme-labelled reagent zone, such that, in use, the substrateis prevented from passing through said binding reagent zone until saidbinding reagent zone is substantially saturated.
 2. The test systemaccording to claim 1 wherein the developing solution and the signalproducing substrate for the enzyme are contained in a rupturable sac inoperating communication with the absorbent material.
 3. The test systemaccording to claim 1, wherein the enzyme is horseradish peroxidase andthe developing solution contains tetramethylbenzidine and hydrogenperoxide.
 4. A test sheet comprising a rigid plastic backing plate orstand with a plurality of systems according to claim 1 attached thereto.